Thromboemolism, a significant medical problem, is manifested by the occlusion of blood vessels due to the presence of blood clots (thrombi) (1). Thrombi are composed of fibrin and blood cells and may form in any part of the cardiovascular system including the veins, arteries, heart and microcirculation (2). As thrombi age, they undergo progressive structural changes; leucocytes are attracted by chemotactic factors released from the aggregated platelets or proteolytic fragments of plasma proteins and become incorporated into the thrombi (3). These aggregated platelets swell and disintegrate and are gradually replaced by fibrin (4). Such clots often affect organs such as the heart and lungs. These clots may loosen and circulate to smaller arteries.
Timely restoration of blood flow through the damaged blood vessels is an important curative goal of medical practices (5). Normal blood flow can be restored by the use of fibrinolytic agents. These agents are called thrombolytic agents, and include streptokinase, other thrombolytic agents, such as urokinase and tissue plasminogen activators are also used in the treatment of myocardial infarction, pulmonary, arterial or venous thromboembolism, surgical adhesions and other cases (6–8,13).
Thrombolytic agents facilitate the in vivo lysis or dissolution of the clot (9). They act by converting endogenous plasminogen (a proenzyme) to plasmin (an active enzyme), which lysis the clot (10–11).
Plaminogen is a single chain glycoprotein, which in its native form has an amino-terminal glutamic acid. It is converted to plasmin by the cleavage of an Arg-Val (560–561) peptide bond (12).
Streptokinase is a single chain polypeptide that binds to plasminogen in a 1:1 ratio. This causes conformational changes so that the complex becomes an active enzyme. This complex cleaves peptide bonds on other plasminogen molecules to produce more plasmin (14).
Bernheimer et al. (1942) (15) have elucidated in detail the media requirements for growth of streptococci. U.S. Pat. No. 2,701,277 describes two primary nitrogenous materials employed in the fementative production of streptokinase by hemolytic streptococci in which the expression of streptokinase is very low (16).
U.S. Pat. No. 3,855,065 describes an improved production process by employing corn steep liquor in the culture medium. However, when streptokinase is prepared from streptococcal cultures grown under these conditions, complex downstream processing is required to remove pyrogenic material such as gram negative bacteria, streptolysin, and other secretory proteins (17).
Streptokinase from Group C streptococci has been purified for clinical usage and studied by several investigators. The primary source of SK has been the culture fluid resulting from the growth of the beta-hemolytic Group C Lansfield streptococcus from a gram negative strain containing other secretory substances which are toxic and pyrogenic to mammals. Extensive purification of the enzyme has become mandatory for its use as a treatment to dissolve thrombi.
Advances in biotechnology have made it possible to excise the streptokinase gene (SK gene) and introduce the gene into other prokaryotic organisms, and thus express the recombinant protein. The host organism, referred to as a recombinant organism, not only has the ability to express the foreign gene, but can also grow to high cell density during fermentation.
German Patent publication IPC C12 N 15/00 describes the cloning and expression of the SK gene from Streptococcus equisimilis strain H46A (ATCC 12449) in an E. coli host and reports a low yield of 0.1 to 1.8 mg/liter (18). Malke et al. (1985) report the nucleotide sequence of native streptokinase from Streptococcus equisimilis H46A (19) (ATCC 12449). U.S. Pat. No. 5,296,366 describes the isolation and cloning of the streptokinase gene under the trp promoter and expressed in E. coli. The transformants produced 350 mg of streptokinase/Liter of medium (20).